Apoptin-associating proteiins

ABSTRACT

The invention relates to the field of apoptosis. The invention provides novel therapeutic possibilities, for example novel combinatorial therapies or novel therapeutic compounds that can work alone, sequentially to, or jointly with apoptin, especially in those cases wherein p53 is (partly) non-functional.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.09/655,109, filed Sep. 5, 2000, pending, the disclosure of which isincorporated herein by this reference in its entirety.

TECHNICAL FIELD

The invention relates to the field of biotechnology, more specificallyto apoptosis, or cell-death.

BACKGROUND OF THE INVENTION

Apoptosis is an active and programmed physiological process foreliminating superfluous, altered or malignant cells (Earnshaw, 1995,Duke et al., 1996). Apoptosis is characterized by shrinkage of cells,segmentation of the nucleus, condensation and cleavage of DNA intodomain-sized fragments, in most cells followed by internucleosomaldegradation. The apoptotic cells fragment into membrane-enclosedapoptotic bodies. Finally, neighboring cells and/or macrophages willrapidly phagocytose these dying cells (Wyllie et al., 1980, White,1996). Cells grown under tissue-culture conditions and cells from tissuematerial can be analyzed for being apoptotic with agents staining DNA,such as DAPI, which stains normal DNA strongly and regularly, whereasapoptotic DNA is stained weakly and/or irregularly (Noteborn et al.,1994, Telford et al., 1992).

The apoptotic process can be initiated by a variety of regulatorystimuli (Wyllie, 1995, White 1996, Levine, 1997). Changes in the cellsurvival rate play an important role in human pathogenesis of diseases,e.g., in cancer development and auto-immune diseases, where enhancedproliferation or decreased cell death is observed (Kerr et al., 1994,Paulovich, 1997). A variety of chemotherapeutic compounds and radiationhave been demonstrated to induce apoptosis in tumor cells, in manyinstances via wild-type p53 protein (Thompson, 1995, Bellamy et al.,1995, Steller, 1995, McDonell et al., 1995).

Many tumors, however, acquire a mutation in p53 during theirdevelopment, often correlating with poor response to cancer therapy.Certain transforming genes of tumorigenic DNA viruses can inactivate p53by directly binding to it (Teodoro, 1997). An example of such an agentis the large T antigen of the tumor DNA virus SV40. For several(leukemic) tumors, a high expression level of the proto-oncogene Bcl-2or Bcr-abl is associated with a strong resistance to variousapoptosis-inducing chemotherapeutic agents (Hockenberry 1994, Sachs andLotem, 1997).

For such tumors lacking functional p53 (representing more than half ofthe tumors) alternative anti-tumor therapies are under development basedon induction of apoptosis independent of p53 (Thompson 1995, Paulovichet al., 1997). One has to search for the factors involved in inductionof apoptosis, which do not need p53 and/or can not be blocked byanti-apoptotic activities, such as Bcl-2 or Bcr-abl-like. These factorsmight be part of a distinct apoptosis pathway or might be (far)downstream of the apoptosis inhibiting compounds.

Apoptin is a small protein derived from chicken anemia virus (CAV;Noteborn and De Boer, 1995, Noteborn et al., 1991, Noteborn et al.,1994; 1998a), which can induce apoptosis in human malignant andtransformed cell lines, but not in untransformed human cell cultures. Invitro, apoptin fails to induce programmed cell death in normal lymphoid,dermal, epidermal, endothelial and smooth-muscle cells. However, whennormal cells are transformed they become susceptible to apoptosis byapoptin. Long-term expression of apoptin in normal human fibroblastsrevealed that apoptin has no toxic or transforming activity in thesecells (Danen-van Oorschot, 1997 and Noteborn, 1996).

In normal cells, apoptin was found predominantly in the cytoplasm,whereas in transformed or malignant cells, i.e., characterized byhyperplasia, metaplasia, dysplasia or aplasia, it was located in thenucleus, suggesting that the localization of apoptin is related to itsactivity (Danen-van Oorschot et al. 1997). Apoptin-induced apoptosisoccurs in the absence of functional p53 (Zhuang et al., 1995a), andcannot be blocked by Bcl-2, Bcr-abl (Zhuang et al., 1995), or theBcl-2-associating protein BAG-1 (Danen-Van Oorschot, 1997a, Noteborn,1996).

Therefore, apoptin is a therapeutic compound for the selectivedestruction of tumor cells, or other hyperplasia, metaplasia, ordysplasia, especially for those tumor cells which have become resistantto (chemo)-therapeutic induction of apoptosis, due to the lack offunctional p53 and (over)-expression of Bcl-2 and otherapoptosis-inhibiting agents (Noteborn and Pietersen, 1998). It appears,that even pre-malignant, minimally transformed cells, are sensitive tothe death-inducing effect of apoptin. In addition, Noteborn and Zhang(1998) have shown that apoptin-induced apoptosis can be used fordiagnosis of cancer-prone cells and treatment of cancer-prone cells.

The fact that apoptin does not induce apoptosis in normal human cells,at least not in vitro, shows that a toxic effect of apoptin treatment invivo will be very low. Noteborn and Pietersen (1998) and Pietersen etal. (1998) have provided evidence that adenovirus expressed apoptin doesnot have an acute toxic effect in vivo. In addition, in nude mice it wasshown that apoptin has a strong anti-tumor activity.

However, to further enlarge the array of therapeutic anti-cancer oranti-auto-immune-disease compounds available in the art, additionaltherapeutic compounds are desired that are designed to work alone,sequentially to, or jointly with apoptin, especially in those caseswherein p53 is (partly) non-functional.

SUMMARY OF THE INVENTION

The invention provides novel therapeutic possibilities, for examplenovel combinational therapies or novel therapeutic compounds that canwork alone, sequentially to, or jointly with apoptin, especially inthose cases wherein p53 is (partly) non-functional.

In a first embodiment, the invention provides an isolated or recombinantnucleic acid or functional equivalent or fragment thereof encoding anapoptin-associating proteinaceous substance capable of providingapoptosis, alone or in combination with other apoptosis inducingsubstances, such as apoptin. Proteinaceous substance herein is definedas a substance comprising a peptide, polypeptide or protein, optionallyhaving been modified by for example glycosylation, myristilation,phosphorylation, the addition of lipids, by homologous or heterologousdi- or multimerization, or any other (posttranslational) modificationsknown in the art.

Apoptin-associating herein is defined as belonging to the cascade ofsubstances specifically involved in the cascade of events found in theapoptosis pathway as inducible by apoptin, preferably those substancesthat are specifically involved in the p53-independent apoptosis pathway.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the partial sequence of vector pMT2SM-AAP-1-a (SEQ IDNO:4). The DNA sequence of the AAP-1-a cDNA is given in bold.

FIG. 2 shows the partial sequence of vector pMT2SM-AAP-1-b. The DNAsequence of the AAP-1-b cDNA is given in bold. (SEQ ID NO:5)

FIG. 3 shows the amino-acid sequence of the analyzed region of theapoptin-associating clone AAP-1-b (bold). In addition, the threeC-terminal amino acids H-E-G of the multiple cloning site of pACT aregiven to illustrate that the AAP-1 amino acid sequence is in frame withthe GAL4-activation domain. (SEQ ID NO:6) This feature proves that theAAP-1 region is indeed synthesized in yeast cells. Note that in FIG. 3amino acid position 23 corresponds with the first amino acid of an AAP-1like protein. Functional domains or fragments herein can for example beidentified as a transcription factor binding domain running from aminoacid position 1 (=23 in FIG. 3) to about 54; a zinc-finger motive,protein-protein interaction and/or protein-nucleic acid interactiondomain running from about amino acid position 25 (=47 in FIG. 3) toabout 42; an apoptosis associated region running from about amino acidposition 32 to 226; a nuclear localization signal running from aboutamino acid position 74 to 81; and a nuclear localization signal runningfrom about amino acid position 102 to 108, or at equivalent positions inanother AAP-1 like protein.

FIG. 4 shows the apoptotic activity of AAP-1-b protein in Saos-2 cells,when expressed alone (filled square) or in combination with apoptin(open square). The percentage of apoptin-induced apoptosis is alsoindicated (filled triangle).

DETAILED DESCRIPTION OF THE INVENTION

In a preferred embodiment, the invention provides an isolated orrecombinant nucleic acid or functional equivalent or fragment thereofencoding an apoptin-associating proteinaceous substance capable ofproviding apoptosis. More preferably the encoded apoptin-associatedproteinaceous substance co-localizes with other apoptosis inducingsubstances, for example apoptin, when the two apoptosis inducingsubstances are present in the same cell. In normal non-transformed cellsthe two apoptosis inducing proteins co-localize in the cytoplasm whereasin transformed or malignant cells the two apoptosis-inducing proteinsco-localizes in the nucleus. In another embodiment theapoptin-associating substance is capable of binding to the mousetranscription factor YY1, which was earlier shown to bind to the mouseAAP-1 homologue RYBP (Garcia et al., 1999). In a most preferredembodiment the isolated or recombinant nucleic acid or functionalequivalent or fragment thereof encoding an apoptin-associatingproteinaceous substance capable of providing apoptosis is derived from acDNA library, preferably a vertebrate cDNA library, such as derivablefrom poultry, but more preferably a mammalian cDNA library, preferablywherein the cDNA library comprises human cDNA.

In another embodiment, the invention provides an isolated or recombinantnucleic acid or functional equivalent or fragment thereof encoding anapoptin-associating proteinaceous substance capable of providingapoptosis capable of hybridizing to a nucleic acid molecule encoding anapoptin-associating proteinaceous substance capable of providingapoptosis as shown in FIG. 1 or 2, in particular encoding a novelprotein capable of providing apoptosis or functional equivalent orfunctional fragment thereof called apoptin-associating protein 1,abbreviated herein also as AAP-1. Of course, an isolated or recombinantnucleic acid or functional equivalent or fragment thereof encoding anadditional apoptin-associating proteinaceous substance capable ofassociating with the AAP-1 protein are herewith also provided, means andmethods to arrive at such an additional protein located in the apoptincascade follow those of the detailed description given herein.

In particular, the invention provides an isolated or recombinant nucleicacid or functional equivalent or fragment thereof encoding anapoptin-associating proteinaceous substance capable of providingapoptosis being at least 70% homologous, preferably at least 80%, morepreferably at least 90%, most preferably at least 95% homologous to anucleic acid molecule, or to a functional equivalent or functionalfragment thereof, encoding an apoptin-associating proteinaceoussubstance as shown in FIG. 1 or 2.

Furthermore, the invention provides a vector comprising a nucleic acidaccording to the invention. Examples of such a vector are given in thedetailed description given herein; such as vector pMT2SM-AAP-1-a or b,pMT2SM vector expressing Myc-tagged AAP-1-a or AAP-1-b cDNAs, a plasmidexpressing an apoptin-associating protein fragment, E. colioverexpression vectors such as pMAL and pET22b comprising a nucleic acidaccording to the invention and so on. These and other vectors are forexample useful in finding additional apoptin-associating proteinaceoussubstances from the cascade, as defined above, or for the(over)expression of a protein encoded by a nucleic acid according to theinvention.

In yet another embodiment, the invention provides a vector comprising anucleic acid according to the invention, the vector comprising agene-delivery vehicle, making the invention very useful in gene therapy.By equipping a gene delivery vehicle with a nucleic acid according tothe invention, and by targeting the vehicle to a cell or cells that havebeen over-proliferating and/or have shown decreased death rates, thegene delivery vehicle provides the cell or cells with the necessarymeans for apoptosis, providing far reaching therapeutic possibilities.

Furthermore, the invention provides a host cell comprising a nucleicacid or a vector according to the invention. Examples comprisetransformed or transfected bacterial or yeast cells as described in thedetailed description herein. Preferred is a host cell according to theinvention which is a transformed eukaryotic cell such as a yeast cell ora vertebrate cell, such as mammalian or Cos cells transformed ortransfected with a nucleic acid or vector according to the invention.The cells are in general capable to express or produce a proteinaceoussubstance capable of providing apoptosis with the ability to associatewith apoptin.

The invention furthermore provides an isolated or recombinantapoptin-associating proteinaceous substance capable of providingapoptosis. As for example shown herein in FIG. 4, expression of suchapoptin-associating proteinaceous substance in cells, such as tumorcells, or other over-proliferating cells, induces the apoptic process.It can do so alone, or in the presence of other apoptosis inducingsubstances such as apoptin, and especially so independent of p53,showing that also in those cases where (functional) p53 is absentapoptosis can be induced by a substance according to the invention. Whenthe apoptin-associated proteinaceous substance capable of providingapoptosis is used together with another apoptosis inducing substance,for example apoptin, the two proteinaceous substances co-localize in thecytoplasm of normal cells and do not result in apoptosis. Whereas intransformed or malignant cells the two apoptosis-inducing proteinsco-localize in the nucleus and induce apoptosis. The invention alsoprovides a proteinaceous substance according to the invention whichbinds to the transcription factor YY1, which was already shown to bindthe AAP-1 mouse homologue RYBP. In particular, the invention provides aproteinaceous substance according to the invention encoded by a nucleicacid according to the invention, for example comprising at least a partof an amino acid sequence as shown in FIG. 3 or a functional equivalentor functional fragment thereof capable of providing apoptosis alone orin combination with other apoptosis inducing substances such as apoptin.

The invention also provides an isolated or synthetic antibodyspecifically recognizing a proteinaceous substance or functionalequivalent or functional fragment thereof according to the invention.Such an antibody is for example obtainable by immunizing an experimentalanimal with a apoptin-associating proteinaceous substance or animmunogenic fragment or equivalent thereof and harvesting polyclonalantibodies from the immunized animal (as shown herein in the detaileddescription), or obtainable by other methods known in the art such as byproducing monoclonal antibodies, or (single chain) antibodies or bindingproteins expressed from recombinant nucleic acid derived from a nucleicacid library, for example obtainable via phage display techniques.

With such an antibody, the invention also provides a proteinaceoussubstance specifically recognizable by such an antibody according to theinvention, for example obtainable via immunoprecipitation, WesternBlotting, or other immunological techniques known in the art.

Furthermore, the invention provides use of a nucleic acid, vector, hostcell, or proteinaceous substance according to the invention for theinduction of apoptosis, as for example shown in FIG. 4. In particular,such use is provided wherein apoptosis is p53-independent. Inparticular, such use is also provided further comprising use of anucleic acid encoding apoptin or a functional equivalent or fragmentthereof or use of apoptin or a functional equivalent or fragmentthereof. As can be seen from FIG. 4, combining these apoptin-inducingsubstances increases the percentage apoptosis of treated tumor cells.

Such use as provided by the invention is particularly useful from atherapeutic viewpoint. The invention provides herewith a pharmaceuticalcomposition comprising a nucleic acid, vector, host cell, orproteinaceous substance according to the invention. In addition, such apharmaceutical composition according to the invention further comprisesa nucleic acid encoding apoptin or a functional equivalent or fragmentthereof or apoptin or a functional equivalent or fragment thereof.

Such a pharmaceutical composition is in particular provided for theinduction of apoptosis, for example wherein apoptosis isp53-independent, for the treatment of a disease where enhanced cellproliferation or decreased cell death is observed, as is in general thecase when the disease comprises cancer or auto-immune disease. Herewiththe invention provides a method for treating an individual carrying adisease where enhanced cell proliferation or decreased cell death isobserved comprising treating the individual with a pharmaceuticalcomposition according to the invention. In particular these compositionscomprise a factor of an apoptosis pathway, which is specific fortransformed cells. Therefore, these compositions are essential for newtreatments, but also for diagnosis of diseases related with aberranciesin the apoptotic process, such as cancer and auto-immune diseases.

In the field of diagnosis the invention provides a method for detectingthe presence of cancer cells or cells that are cancer prone in a sampleof cells comprising transfecting cells in the sample with a nucleic acidor a vector according to the invention culturing the cells anddetermining the percentage of apoptosis of cells in the sample. Forexample, we can conclude that the cellular localization of AAP-1 isdifferent in tumorigenic/transformed cells in comparison to normalnon-transformed cells. Furthermore, accumulation of AAP-1 in the nucleuscorrelates with apoptosis induction, whereas cytoplasmic localizationcorrelates with cell viability and normal proliferative capacity. Theinvention thus provides a method for detecting the presence of cancercells or cells that are cancer prone in a sample of cells comprisingtransfecting cells in the sample with a nucleic acid or a vectoraccording to the invention and determining the intracellularlocalization of a proteinaceous substance derived from the nucleic acidor vector in cells in the sample. In particular, the invention providesa method wherein the presence of the proteinaceous substance in thecells is detected by immunostaining the cells with an antibody, such aswith an immunofluorescence assay, or another immunoassay known in theart. Preferably, the antibody comprises an antibody according to theinvention.

Also, the invention provides a method for identifying a putativecancer-inducing agent, such as transforming genes or functionalfragments thereof comprising exposing a sample of cells to the agent,for example by transfection, or by merely providing the agent to themedium surrounding the cells, and detecting the presence of cancer cellsor cells that are cancer prone in a sample of cells with a methodaccording to the invention.

In addition, the invention provides a method to detect cancer-pronenessof a sample of cells, and thereby to detect cancer-proneness of theindividual from which those cells were sampled, comprising submittingthe cells to a cancer-inducing agent, such as UV-light, and detectingthe presence of cancer cells or cells that are cancer prone in a sampleof cells with a method according to the invention.

The invention will be explained in more detail in the following detaileddescription which does not limit the invention.

Experimental

We have used the yeast-2 hybrid system (Durfee et al., 1993) to identifyapoptin-associating cellular compounds, which are essential in theinduction of apoptosis. The system uses an in vivo strategy to identifyhuman proteins capable of physically associating with apoptin. It hasbeen used to screen cDNA libraries for clones encoding proteins capableof binding to a protein of interest (Fields and Song, 1989, Yang et al.,1992). The invention provides, for example, a novel apoptin-associatingprotein, one of which is named apoptin-associating protein 1,abbreviated as AAP-1. The invention also provides a method for inducingapoptosis through interference with the function of this newlydiscovered AAP-1 protein or functional equivalents or fragments thereofand/or the induction of apoptosis by means of (over)expression of AAP-1or related gene or functional equivalents or fragments thereof.

The invention also provides an anti-tumor therapy based on theinterference with the function of AAP-1-like proteins and/or its(over)expression. AAP-1-like proteins are normally not very abundant inimmortalized cell lines. Therefore, an aberrant high level of AAP-1-likeproteins will result in the induction of the opposite process of celltransformation, namely apoptosis. The invention furthermore provides themediator of apoptin-induced apoptosis, which is tumor-specific. Theinvention provides a therapy for cancer, auto-immune diseases or relateddiseases which is based on AAP-1-like proteins alone or in combinationwith apoptin and/or apoptin-like compounds.

Materials and Methods

Construction of pGBT9-VP3

For the construction of the bait plasmid, which enables theidentification of apoptin-associating proteins by means of ayeast-two-hybrid system, plasmid pET-16b-VP3 (Noteborn unpublishedresults) was treated with NdeI and BamHI. The 0.4 kb NdeI BamHI DNAfragment was isolated from low-melting-point agarose. GAL4-activationdomain-tagged cDNA library.

The expression vector pACT, containing the cDNAs fromEpstein-Barr-virus-transformed human B cells fused to the GAL4transcriptional activation domain, was used for detectingapoptin-associating proteins. The pACT cDNA library is derived from thelambda-ACT cDNA library, as described by Durfee et al. 1993.

Bacterial and Yeast Strains

The E. coli strain JM109 was the transformation recipient for theplasmid pGBT9 and pGBT-VP3. The bacterial strain electromax/DH10B wasused for the transformation needed for the recovery of theapoptin-associating pACT-cDNAs, and was obtained from GIBCO-BRL, USA.

The yeast strain Y190 was used for screening the cDNA library, and allother transformations, which are part of the yeast-two-hybrid system.

Media

For drug selections Luria Broth (LB) plates for E. coli weresupplemented with ampicillin (50 microgram per ml). Yeast YPD and SCmedia were prepared as described by Rose et al. (1990).

Transformation of competent yeast strain Y190 with plasmids pGBT-VP3 andpACT-cDNA and screening for beta-galactosidase activity.

The yeast strain Y190 was made competent and transformed according tothe methods described by Klebe et al. (1983). The yeast cells were firsttransformed with pGBT-VP3 and subsequently transformed with pACT-cDNA,and these transformed yeast cells were grown on histidine-minus plates,also lacking leucine and tryptophan.

Hybond-N filters were laid on yeast colonies, which werehistidine-positive and allowed to wet completely. The filters werelifted and submerged in liquid nitrogen to permeabilize the yeast cells.The filters were thawed and placed with the colony side up on Whattman 3MM paper in a petri dish with Z-buffer (Per liter: 16.1 gr Na₂HPO₄.7H₂O,5.5 gr NaH₂PO₄.H₂O, 0.75 gr KCl and 0.246 gr MgSO₄.7H₂O, pH 7.0)containing 0.27% beta-mercapto-ethanol and 1 mg/ml X-gal. The filterswere incubated for at least 15 minutes or during night.

Recovery of Plasmids from Yeast

Total DNA from yeast cells, which were histidine- andbeta-galactosidase-positive, was prepared by using theglusulase-alkaline lysis method as described by Hoffman and Winston(1987) and used to transform Electromax/DH10B bacteria viaelectroporation using a Bio-Rad GenePulser according the manufacturer'sspecifications.

Transformants were plated on LB media containing the antibiotic agentampicillin.

Isolation of Apoptin-Associating pACT Clones

By means of colony-filter assay the colonies were lysed and hybridizedto a radioactive-labeled 17-mer oligomer, which is specific for pACT(see also section Sequence analysis). Plasmid DNA was isolated from thepACT-clones, and by means of XhoI digestion analyzed for the presence ofa cDNA insert.

Sequence Analysis

The subclones containing the sequences encoding apoptin-associatingproteins were sequenced using dideoxy NTPs according to theSanger-method, which was performed by Eurogentec, Seraing, Belgium). Theused sequencing primer was a pACT-specific 17-mer comprising of theDNA-sequence 5′ TACCACTACAATGGATG-3′. (SEQ ID NO:1)

The sequences of the apoptin-associating cDNAs were compared with knowngene sequences from EMBL/Genbank.

Construction of pMAL-AAP-1 and pET22b-AAP-1

For the construction of the protein overexpression plasmids, whichenable the production and isolation of apoptin-associated protein,plasmids pMALTB and pET22b were used. Plasmid pMALTB is a derivative ofpMAL-C2 (New England Biolabs) in which the factor Xa site has beenreplaced by a Trombin site. Plasmid pMALTB was treated with BamHI andSalI and the ±7.0 kb DNA fragment was isolated from an agarose/TBE gel(QIAGEN® gel extraction kit). The AAP-1 sequence encoding the completeopen reading frame was obtained by a PCR reaction on pACT-AAP-lb withthe forward primer: 5′-AACGGGATCCGGCGGCATGGGCGACAAGAAGAGCCCGACC-3′ (SEQID NO:7) and the reversed primer:5′-AAAAGTCGACTCAGAAAGATTCATCATTGACTGCTGACAT-3′; (SEQ ID NO:8) the ±0.7kb PCR fragment was digested with BamHI and SalI and isolated from anagarose/TBE gel (QIAGEN® gel extraction kit). The final constructcontaining a fusion between the MBP gene and the AAP-1 gene under theregulation of the IPTG inducible tac promoter was called pMAL-AAP-1.

Plasmid pET22b (Novagen) was treated with NdeI and NotI and the ±5.5 kbDNA fragment was isolated from an agarose/TBE gel (QIAGEN® gelextraction kit). The AAP-1 sequence encoding the open reading frame wasobtained by a PCR reaction on pACT-AAP-lb with the forward primer:5′-GGGAATTCCATATGGGCGACAAGAAGAG CCCGACC-3′ (SEQ ID NO:9) and thereversed primer: 5′-AAGGAAGTACGCGGCCGCGAAAGATTCATCATTGACTGCTGACATGT-3′;(SEQ ID NO:10) the PCR product was treated with NdeI and NotI and the±0.7 kb fragment was isolated from an agarose/TBE gel (QIAGEN® gelextraction kit). The final construct containing a fusion between theAAP-1 gene and the (His)6-tail under the regulation of the IPTGinducible T7lac promoter was called pET22b-AAP-1.

Both constructs were proven to be correct by restriction-enzyme analysisand DNA-sequencing according to the Sanger method (1977).

All cloning steps were essentially carried out as described by Maniatiset al. (1992).

Bacterial Strains for Overexpression of MBP-AAPI and AAP-1-(His)6

For protein production with the plasmid pMAL-AAP-1 the E. coli strainB834(λDE3) was used and for the plasmid pET22b-AAP-1 the E. coli strainBL21(DE3) was used. Both strains were obtained from Novagen.

Apoptin induces specifically apoptosis in transformed cells, such ascell lines derived from human tumors. To identify the essentialcompounds in this cell-transformation-specific and/or tumor-specificapoptosis pathway, a yeast genetic screen was carried out.

We have used a human cDNA library, which is based on the plasmid vectorpACT containing the complete cDNA copies made from Epstein-Barrvirus-transformed human B cells (Durfee et al., 1993).

The following examples are offered by way of illustration of the presentinvention, not limitation.

EXAMPLE 1 Construction of a Bait Plasmid Expressing a Fusion GeneProduct of GAL4-DNA-Binding Domain and Apoptin

To examine the existence of apoptin-associating proteins in the humantransformed/tumorigenic cDNA library, a so-called bait plasmid had to beconstructed. To that end, the complete apoptin-encoding region, flankedby about 40 basepairs downstream from the apoptin gene, was cloned inthe multiple cloning site of plasmid pGBT9.

The final construct, called pGBT-VP3, was analyzed by restriction-enzymeanalysis and sequencing of the fusion area between apoptin and theGAL4-DNA-binding domain.

EXAMPLE 2 A Gene (Fragment) Encoding an Apoptin-Associating Protein isDetermined by Transactivation of a GAL4-Responsive Promoter in Yeast

The apoptin gene is fused to the GAL4-DNA-binding domain of plasmidpGBT-VP3, whereas all cDNAs derived from the transformed human B cellsare fused to the GAL4-activation domain of plasmid pACT. If one of theproteinaceous substances encoded by the cDNAs binds to apoptin, theGAL4-DNA-binding domain will be in the vicinity of the GAL4-activationdomain resulting in the activation of the GAL4-responsive promoter,which regulates the reporter genes HIS3 and LacZ.

The yeast clones containing plasmid expressing apoptin and a plasmidexpressing an apoptin-associating protein fragment can grow on ahistidine-minus medium and will stain blue in a beta-galactosidaseassay. Subsequently, the plasmid with the cDNA insert encoding theapoptin-associating protein can be isolated and characterized.

Before we could do so, however, we have determined that transformationof yeast cells with pGBT-VP3 plasmid alone, or in combination with anempty pACT vector, did not result in the activation of theGAL4-responsive promoter.

EXAMPLE 3 Identification of Apoptin-Associating Proteins Encoded bycDNAs Derived from a Human Transformed B Cell Line

We have found two yeast colonies, which upon transformation withpGBT-VP3 and pACT-cDNA were able to grow on histidine-minus medium (alsolacking leucine and tryptophan) and stained blue in a beta-galactosidaseassay. These results indicate that the observed yeast colonies contain,besides the bait plasmid pGBT-VP3, also a pACT plasmid encoding apotential apoptin-associating protein.

Plasmid DNA was isolated from the positive yeast colony, which wastransformed in bacteria. By means of a filter-hybridization assay usinga pACT-specific labeled DNA-probe, the clones containing pACT plasmidcould be determined. Subsequently, pACT DNA was isolated and digestedwith restriction enzyme XhoI, which is indicative for the presence of acDNA insert. Finally, the pACT plasmids containing a cDNA insert wassequenced by using the Sanger method (Sanger et al., 1977).

EXAMPLE 4 Description of Apoptin-Associating Proteins

The yeast genetic screen for apoptin-associating proteins resulted inthe detection of two cDNA clones A and B comprising a single type ofprotein, namely a novel protein called apoptin-associating protein 1,abbreviated as AAP-l. The cDNA AAP-l-b (SEQ. ID NO:5) harbors thecomplete open reading frame with an ATG-initiation codon, whereas theAAP-l-a (SEQ. ID NO:4) cDNA sequence contains a partial AAP-1 openreading frame, which is completely homologous to the AAP-1-b DNAsequence.

The determined DNA sequence of the AAP-l-a and AAP-1-b cDNA clones areshown in FIGS. 1 and 2, respectively. The amino acid sequence, derivedfrom the detected DNA sequence of clone AAP-1-b, which represents thecomplete AAP-1a.a. sequence, is given in FIG. 3 (SEQ. ID NO:6).

EXAMPLE 5 Construction of an Expression Vector for the Identification ofAAP-1 Protein in Mammalian Cells

To study whether the cloned cDNAs AAP-1-a and AAP-1-b indeed encode(apoptin-associating) protein products, we have carried out thefollowing experiments.

The DNA plasmid pMT2SM contains the adenovirus 5 major late promoter(MLP) and the SV40 ori enabling high levels of expression of foreigngenes in transformed mammalian cells, such as SV-40-transformed Coscells.

Furthermore, the pMT2SM vector contains a Myc-tag (amino acids:EQKLISEEDL) (SEQ ID NO:2) which is in frame with the foreign-geneproduct. This Myc-tag enables the recognition of, e.g.,apoptin-associating proteins by means of the Myc-tag-specific 9E10antibody.

The pMT2SM vectors expressing Myc-tagged AAP-1-a or AAP-1-b cDNAs wereconstructed as follows. The pACT-AAP-l-a and pACT-AAP-1-b cDNA cloneswere digested with the restriction enzyme XhoI and the cDNA inserts wereisolated. The expression vector pMT2SM was digested with XhoI andtreated with calf intestine alkaline phosphatase and ligated to theisolated AAP-l cDNA inserts. By sequence analysis, the pMT2SM constructscontaining the AAP-1-a or AAP-1-b cDNA in the correct orientation wereidentified.

The synthesis of Myc-tagged AAP-1 protein was analyzed by transfectionof Cos cells with plasmid pMT2SM-AAP-1-a or pMT2SM-AAP-1-b. As negativecontrol, Cos cells were mock-transfected. Two days after transfection,the cells were lysed and Western-blot analysis was carried out using theMyc-tag-specific antibody 9E10.

The Cos cells transfected with pMT2SM-AAP-1-a and pMT2SM-AAP-1-b wereproven to synthesize a specific Myc-tagged AAP-1 product with theexpected size of approximately 33 kDa (AAP-1-a) or 35 kDA (AAP-1-b). Asexpected, the lysates of the mock-transfected Cos cells did not containa protein product reacting with the Myc-tag-specific antibodies.

These results indicate that we have been able to isolate cDNAs that areable to produce a protein product with the ability to associate to theapoptosis-inducing protein apoptin.

EXAMPLE 6 Co-Immunoprecipitation of Myc-Tagged AAP-1 Protein withApoptin in a Transformed Mammalian Cell System

Next, we have analyzed the association of apoptin and the AAP-1 proteinby means of co-immunoprecipitations using the Myc-tag-specific antibody9E10. The 9E10 antibodies were shown not to bind directly to apoptin,which enables the use of 9E10 for carrying out co-immuno-precipitationswith (myc-tagged) apoptin-associating proteins and apoptin.

To that end, Cos cells were co-transfected with plasmid pCMV-VP3encoding apoptin and with plasmid pMT2SM-AAP-1-a. As a negative control,cells were transfected with pCMV-VP3 expressing apoptin and a plasmidpcDNA3. 1. LacZ-myc/His-LacZ encoding the myc-tagged beta-galactosidase,which does not associate with apoptin.

Two days after transfection, the cells were lysed in a buffer consistingof 50 mM Tris (PH 7.5), 250 mM NaCl, 5 mM EDTA, 0.1% TRITON X100, 1mg/ml Na₄P₂O₇ and freshly added protease inhibitors such as PMSF,Trypsine-inhibitor, Leupeptine and Na₃VO₄. The specific proteins wereimmuno-precipitated as described by Noteborn et al. (1998) using theMyc-tag-specific antibodies 9E10, and analyzed by Western blotting.

Staining of the Western blot with 9E10 antibodies and 111.3 antibodies,which are specifically directed against myc-tag and apoptin,respectively, showed that the “total” cell lysates contained apoptin andthe Myc-tagged AAP-1 protein or beta-galactosidase product.Immunoprecipitation of the Myc-tagged AAP-1 products was accompanied bythe immuno-precipitation of apoptin product of 16 kDa. In contrast,immunoprecipitation of myc-tagged beta-galactosidase did not result in asignificant co-precipitation of the apoptin protein.

In total, three independent immunoprecipitation experiments were carriedout, which all showed the associating ability of apoptin to the AAP-1protein.

These results indicate that the novel determined AAP-1 protein is ableto specifically associate with apoptin not only in the yeast background,but also in a mammalian transformed cellular system.

EXAMPLE 7 Over-Expression of the Novel AAP-1 Protein in HumanTransformed Cells Induces the Apoptotic Process

In addition, we have examined whether AAP-1 carries apoptotic activity.First, we have analyzed the cellular localization of the novel AAP-1protein in human transformed cells. To that end, the humanosteosarcoma-derived Saos-2 cells were transfected, as described byDanen-van Oorschot (1997), with plasmid pMT2SM-AAP-l-a or pMT2SM-AAP-1-bencoding the myc-tagged AAP-1-a or AAP-1-b protein, respectively.

By indirect immunofluorescence using the myc-tag-specific antibody 9E10and DAPI, which stains the nuclear DNA, it was shown that both thepartial and complete AAP-1 protein were present in the nucleus of thecell. Actually, it co-localizes with the chromatin/DNA structures.

Finally, we examined whether (over)-expression of both cDNAs encodingcomplete or partial AAP-1 protein results in induction of apoptosis.Four days after transfection, the majority of AAP-1-positive cells wereaberrantly stained with DAPI, which is indicative for induction ofapoptosis (Telford, 1992, Danen-van Oorschot, 1997).

Co-expression of apoptin and both AAP-l proteins in human tumor cells,such as Saos-2 cells, results in an apoptotic process faster thanexpression of apoptin or AAP-1 protein alone. The results of theapoptotic activity of the complete AAP-1 protein are shown in FIG. 4.

The fact that AAP-1 protein can induce apoptosis in p53-minus Saos-2cells indicates that AAP-1 can induce p53-independent apoptosis. Theseresults imply that AAP-1 can be used as an anti-tumor agent in caseswhere other (chemo)therapeutic agents will fail. Furthermore, thefinding that both apoptin and AAP-1 induce a p53-independent pathwayindicates that AAP-1 fits in the apoptin-induced apoptotic pathway.

EXAMPLE 8 Co-Localization of Apoptin and AAP-l in Human Tumor Cells

To establish the possible co-localization of apoptin and AAP-l intransformed human cells, plasmids encoding apoptin and AAP-1 weretransfected in Saos-2 cells. Expression of AAP-1 and apoptin wasmonitored by indirect immunofluorescence by means of a confocal-laserscanning microscopy with the use of specific antibodies mAb myc 9E10against the myc-tag on AAP-1 and pAb VP3-c against the C-terminus ofapoptin.

Cells co-transfected with a plasmid encoding AAP-1 and a plasmidencoding apoptin expressed these proteins predominantly in the nucleus.Both, apoptin and AAP-1 had granular structures and the above describedcharacteristic structures. By means of confocal-laser scanningmicroscopy, partial co-localization of AAP-1 and apoptin was clearlyshown to occur in these nuclear structures.

In conclusion, we have identified an apoptin-associating protein, namelythe novel AAP-1 protein, which is present in the nucleus and able toinduce (p53-independent) apoptosis in human tumor cells. Furthermore,when AAP-1 and apoptin are expressed in the same cell the two apoptosisinducing proteins are co-localizing in the nucleus of human tumor cells.

EXAMPLE 9 AAP-1 Localizes in Human Normal Diploid Cells in CytoplasmicStructures

Next, we have examined whether AAP-1 behaves similar in normal humandiploid non-transformed cells as has been found for AAP-1 in human tumorcells.

To that end, human diploid VH10 fibroblasts (Danen-Van Oorschot, 1997)were transfected using FUGENE® according the protocol of the supplier(Roche, Almere, The Netherlands), with plasmid pMT2SM-AAP-lb encodingthe myc-tagged complete AAP protein. In parallel, human tumor-derivedSaos-2 cells were also transfected with plasmid pMT2SM-AAP-1-b.

By indirect immunofluorescence using the myc-tag-specific antibody 9E10,it was shown that in normal diploid VH10 fibroblasts AAP-1 protein islocated in the cytoplasm. As expected, in the human tumor Saos-2 cellsAAP-l is located in the nucleus.

Furthermore, we have examined the effect of co-expression of AAP-l andapoptin in human VH10 fibroblasts on the cellular localization of AAP-l,as described for cells expressing AAP-1 alone. The immunofluorescencedata show that both apoptin and AAP-1 are located in cytoplasmicstructures. These findings indicate that AAP-l and/or apoptin expressiondo not result in the nuclear localization of one or the other in normal(human) diploid cells.

EXAMPLE 9 Co-Localization of Apoptin and AAP-l in Human Fibroblasts

To establish the possible co-localization of apoptin and AAP-l innon-transformed cells, plasmids encoding apoptin and AAP-1 weretransfected in VH10 cells. Expression of AAP-l and apoptin weremonitored by indirect immunofluorescence by means of a confocal-laserscanning microscopy with the use of specific antibodies mAb myc 9E10against the myc-tag on AAP-l and pAb VP3-c against the C-terminus ofapoptin. Cells were screened for (co-) localization of apoptin and AAP-1and for induction of apoptosis by nuclear staining with DAPI.

Cells co-transfected with a plasmid encoding AAP-l and a plasmidencoding apoptin expressed these proteins predominantly in thecytoplasm. Apoptin and AAP-1 both had a thready and sometimes granularstructure. By means of confocal-laser scanning microscopy,co-localization of AAP-1 and apoptin was clearly shown to occur in thesecytoplasmic structures. Similar cytoplasmic structures were observedwith AAP-l, when expressed alone or vice versa when apoptin wasexpressed alone.

Thus, in the presence or absence of apoptin, AAP-l has a cytoplasmiclocalization and thready-aggregated structures in non-transformed humanfibroblasts.

In conclusion, we have identified an apoptin-associating protein, namelyAAP-1, which is differentially located in human diploid non-transformedcells versus human tumorigenic cells. These results show that AAP-lfunctions in a different way in normal diploid cells as in tumorigeniccells. Furthermore, when ADP-1 and apoptin are expressed in the samenormal non-transformed cell the two apoptosis inducing proteins areco-localizing in the cytoplasm.

EXAMPLE 10 The Effect on Induction of Covalent Linkage of an SV40 LargeT Antigen Nuclear Localization Signal to the Apoptin Protein

In the following experiments, we have examined whether expression of achimeric protein consisting of apoptin and the nuclear localizationsignal of SV40 LT antigen (amino acidsN-terminal-Proline-Proline-Lysine-Lysine-Lysine-Arginine-Lysine-Valine—C-terminal (SEQ. ID NO:7) of SV40 large T antigen covalently linked tothe N-terminus of apoptin) results in the induction of apoptosis innon-transferred and transformed human cells. The chimeric protein iscalled NLS-apoptin.

To that end, non-transformed VH10 human fibroblasts and transformedhuman osteosarcoma-derived Saos-2 cells (Danen-van Oorschot et al.,1997) were transfected with a plasmid encoding the chimeric proteinNLS-apoptin. In transformed human cells, expression of NLS-apoptinresulted in the nuclear localization of apoptin and induction ofapoptosis. Expression of NLS-apoptin in normal human fibroblasts,however, resulted in the nuclear localization of apoptin, but not ininduction of apoptosis. This indicates that “forcing” of transportingapoptin into the nucleus does not result in its apoptotic activity perse. Apoptin seems to require an additional tumor-related event.

EXAMPLE 11 The Effect of Expression of NLS-Apoptin on AAP-1 in NormalHuman Fibroblasts

Next, we have examined whether the expression of NLS-apoptin caninfluence the cellular location of AAP-1 and/or its apoptotic activityin normal human fibroblasts. To that end, VH10 cells were co-transfectedwith plasmids encoding NLS-apoptin or AAP-1. By means ofindirect-immunofluorescence and DAPI-staining using a fluorescencemicroscopy, it was clearly established that both NLS-apoptin and AAP-1were located in the nucleus without inducing apoptosis. This indicatesthat “forcing” of transporting AAP-1 through NLS-apoptin into thenucleus does not result in AAP-l apoptotic activity per se. AAP-l likeapoptin seems to require an additional tumor-related event to becomeapoptotic in the nucleus of transformed cells.

EXAMPLE 12 AAP-l does not Induce Apoptosis in Human DiploidNon-Transformed Cells

In the following experiments we have examined whether AAP-l alone or incombination with apoptin is also able to induce apoptosis in humandiploid non-transformed fibroblasts as has been observed for humantumorigenic/transformed cells.

To that end, VH10 cells were transfected with plasmid pMT2SM-AAP-l-b asdescribed above. The transfected cells were analyzed by indirectimmunofluorescence using the myc-tag-specific and/or apoptin-specificantibodies and DAPI-staining. DAPI stains intact DNA in a different waythat apoptotic DNA (Telford et al., 1992). The analysis clearly showsthat VH10 fibroblasts containing AAP-1 protein alone or both AAP-1 andapoptin do not undergo apoptosis.

The obtained results show that apoptin-related proteins such as AAP-1might behave in a different way in “healthy” cells in comparison totumor cells.

EXAMPLE 13 Diagnostic Assay for Cancer Cells

Based on the present report, we can conclude that the cellularlocalization of AAP-1 is different in tumorigenic/transformed humancells in comparison to normal human non-transformed cells. Furthermore,accumulation of AAP-1 in the nucleus correlates with apoptosisinduction, whereas cytoplasmic localization correlates with cellviability and normal proliferative capacity. Therefore, we are able todevelop a diagnostic assay for the identification of (human) cancercells versus normal “healthy” non-transformed cells.

The assay consists of transfecting “suspicious” (human) cells, forinstance from human origin, with a plasmid encoding AAP-1, or infectingthe cells with viral vectors expressing AAP-1. Subsequently, the cellswill be examined, 1) for the ability to undergo apoptosis by theover-expressing AAP-l gene and 2) for a shift in the localization ofAAP-1 from the cytoplasm to the nucleus.

The intracellular localization of AAP-1 can be determined, using animmunofluorescence assay with monoclonal antibodies specific for AAP-1and/or specific for a tag linked to AAP-1 such as the herein describedmyc-tag. If the percentage of apoptosis and/or the nuclear localizationof AAP-1 in the analyzed cells expressing AAP-1 is significantly higherthan in AAP-1-positive control “healthy” cells, one can conclude thatthe analyzed cells have become tumorigenic/transformed. As a positivecontrol, known human tumorigenic cells will be used for expressingAAP-1.

EXAMPLE 14 Co-Expression of SV40 Large T Antigen and AAP-1 Results inTranslocation of AAP-1 and Induction of Apoptosis

We have examined the effect of expression of transforming genes onAAP-1-induced apoptosis in normal human cells derived from healthyindividuals. To that end, human VH10 diploid fibroblasts weretransiently co-transfected with plasmid pMT2SM-AAP-1-b encoding thecomplete AAP-l protein and either plasmid pR-s884 encoding SV40 large Tantigen, or the negative-control plasmid pCMV-neo (Noteborn and Zhang,1998).

By indirect immunofluorescence, the cells were analyzed forAAP-l-induced apoptosis. The normal VH10 cells did not undergo apoptosiswhen AAP-l was transfected with the negative-control plasmid. Theresults showed, as expected, that expression of AAP-l is not able toinduce apoptosis in normal human diploid cells, confirming the abovementioned data. However, normal diploid human fibroblasts expressingboth AAP-l and SV40 large T antigen underwent AAP-l-induced apoptosis.

The transition of normal human cells, from AAP-1-resistance toAAP-1-susceptibility, can probably be explained by the fact that theAAP-1 protein translocates from a cytoplasmic localization to a nuclearlocalization. This transition becomes apparent already two days aftertransfection of plasmids encoding the transforming protein SV40 large Tantigen. One can conclude that an event takes place, in this example dueto expression of a transforming product derived from a DNA-tumor virus,which results in the translocation of over-expressed AAP-1 from thecytoplasm to the nucleus, which is followed by induction of apoptosis.

EXAMPLE 15 Diagnostic Assay for Cancer-Inducing Genes, Agents andCancer-Proneness Based on AAP-1-Induced Apoptosis

Based on the present report, we are able to develop a diagnostic assayfor the identification of cancer-inducing and/or transforming agents orgenes.

A first type of assay consists of transfecting “normal” cells, forinstance, from human origin, with a plasmid encoding AAP-1, or infectingthe cells with viral vectors expressing AAP-1, together with a plasmidencoding a putative transforming/cancer-inducing gene. Subsequently, thecells will be examined, 1) for the ability to undergo apoptosis by theover-expressing AAP-1 gene and 2) for a shift in the localization ofAAP-1 from the cytoplasm to the nucleus.

The intracellular localization of AAP-1 can be determined, using animmunofluorescence assay with monoclonal antibodies specific for AAP-1and/or specific for a tag linked to AAP-1 such as the herein describedmyc-tag. If the percentage of apoptosis and/or the nuclear localizationof AAP-1 in normal cells co-expressing AAP-1 and the putativetransforming/cancer-inducing gene is significantly higher than inAAP-1-positive control cells expressing a control plasmid, one canconclude that the analyzed gene indeed has transforming/cancer-inducingactivity.

A second example of a diagnostic test is based on the treatment ofcultured normal diploid cells with a putative carcinogenic agent. Theagent can be added, for instance, to the culture medium for variouslengths of time. Subsequently, the cells are transfected with a plasmidencoding AAP-1. This approach can also be carried out by firsttransfecting/infecting the normal diploid cells, and then treating thecells with the agent to be tested.

The subsequent steps of the assay are the same as described for the inthis section described first type of diagnostic assay. If the percentageof apoptosis and/or the nuclear localization of AAP-1 in normal cellsexpressing AAP-1 and the putative carcinogenic agent is significantlyhigher than in AAP-1-positive control cells expressing a control agent,one can conclude that the analyzed agent indeed hastransforming/cancer-inducing activity.

A third example of a diagnostic test is based on the treatment ofcultured normal diploid cells derived from a skin biopsy of thepotential cancer-prone individual to be tested and cultured in suitablemedium. Next, the cells are irradiated with UV and subsequentlytransfected with a plasmid encoding AAP-1, or infected with a viralvector expressing AAP-1, or the cells are first transfected/infected andthen irradiated. In parallel, diploid cells from a normal healthyindividual will be used as control.

The subsequent steps of the assay are the same as described for thefirst type of diagnostic assay. If after UV-treatment the percentage ofapoptosis and/or the nuclear localization of AAP-1 in diploid cellsderived from the potential cancer-prone individual is significantlyhigher than in UV-treated AAP-1-positive control cells, one can concludethat the analyzed individual is cancer-prone.

EXAMPLE 16 AAP-1 and YY1 Associate with Each Other in Transformed Cells

The mouse homologue of AAP-1, RYBP (Garcia et al., 1999) can interactwith YY1. YY1 can activate or repress the transcription of cellular andviral proteins. Protein-protein interactions can influence the activityof YY1. AAP-1 might interact with YY1. The interaction between AAP-1 andYY1 could influence the activity of YY1, resulting in transcriptionalrepression or activation. Transcription of genes could be important forthe apoptotic activity of apoptin and/or AAP-1.

To establish whether AAP-l forms a complex with YY1 in vitro, plasmidsencoding AAP-l and YY1 (pCMV-HAVY-1; Kind gift of Dr Yang Shi, HarvardMedical School, Boston, USA) were co-transfected in Cos-cells. As anegative control, YY-1 was co-expressed with LacZ. The cells were lysedand an immunoprecipitation assay was performed with the use of specificantibodies against the myc-tag of AAP-l (or LacZ), anti-myc 9E10, andagainst YY1, anti-YY1. The resulting complexes were analyzed byWestern-blotting techniques. In the cell-lysates, AAP-l and/or YY1and/or LacZ were detected, as expected.

In cells transfected with AAP-1 and YY1 it was clearly visible that whenwe performed an immunoprecipitation with antibodies specific formyc-tagged AAP-l, both AAP-1 and YY1 were detectable. The blot showed aband at 32 kDa (AAP-l) and a band at 67 kDa (YY1). Additionally, weperformed an immunoprecipitation using specific antibodies for YY1.Again, both YY1 and AAP-l were detectable on the Western blot.

In cells transfected with a plasmid expressing only AAP-1, only AAP-1could be detected. Cells transfected with a plasmid encoding YY1 showedexpression of YY1, AAP-l was not detected. To rule out a non-specificbinding of AAP-l with YY1, cells were transfected with AAP-1 and LacZand immunoprecipitations were carried out with antibodies specific forLacZ. Although, LacZ protein was clearly visible on the Western Blot, noAAP-l protein was visible.

Therefore, the obtained results show that AAP-1 specifically binds tothe transcription-related factor YY1.

EXAMPLE 17 Production and Isolation of MBP-AAP-1 and AAP-1-(His)6

To examine the possibility of MBP-AAP-1 and AAP-1-(His)₆ fusion proteinproduction the AAP-1 nucleic acid coding for the open reading frame wascloned in the protein overexpression cassettes pMALTB and pET22b.Inducing the E. coli cells according to the manufactures instructions(Novagen) leads to the production of soluble fusion protein.

Isolation of the MBP AAP-1 fusion protein via affinity chromatography(amylose resin, New England Biolabs) and ion exchange chromatography(High-S, BIORAD®) leads to a 90 to 95% pure, full length protein. Sizeexclusion chromatography (AMERSHAM® SUPEROSE®6 HR 10/30) of this proteinshows that a substantial part of the MBP-AAP-1 preparation appears tobehave as one distinct species, a Homotrimer or Homo-tetramer. Thisfinding would suggest that recombinant AAP-1 (in the form of a MBPfusion protein) is capable of assuming a correct fold and is likely tobe biologically active.

Isolation of the AAP-1-(His)₆ protein with metal affinity chromatography(Ni²⁺-NTA, QIAGEN®) leads to a ±80% pure protein batch.

In conclusion, fusion of AAP-1 to MBP results in properly folded,soluble AAP-1 which is likely to be biologically active.

EXAMPLE 18 Production of Polyclonal Antibodies Directed Against AAP-1Proteins

For the production of polyclonal antibodies against AAP-1 proteins aputative immunogenic peptide was synthesized (AAP-1 peptide consists ofthe amino acids N/terminus-CTKTSETNHTSRPRLK-C/terminus; EUROGENTEC® SA,Belgium). (SEQ ID NO:3) Subsequently, rabbits were injected with thespecific peptides according the standard procedures of the manufacturer.

The serum derived from the rabbits injected with the AAP-1 peptide wasshown to be specific for the above described AAP-1 products by means ofELISA and Western-blot assays. These results imply that we havegenerated specific antibodies, which can be used for detecting theapoptin-associating protein AAP-1.

In conclusion, we have provided evidence that interference of specificfactors with the function of AAP-1 proteins results in induction ofapoptosis.

Therapies based on induction of (p53-independent) apoptosis are possibleutilizing the interference with the function of AAP-1 proteins. Anexample of such an interfering factor is apoptin. Another CAV-derivedprotein, which is known to induce apoptosis and also known to enhanceapoptin activity is VP2 (Noteborn et al., 1997).

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporate by reference.

The invention now having been fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

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1.-11. (canceled)
 12. An isolated or recombinant apoptin-associatingproteinaceous substance that induces apoptosis and comprises amino acids54 to 248 of SEQ ID NO:6.
 13. The isolated or recombinantapoptin-associating proteinaceous substance of claim 12 wherein saidisolated or recombinant apoptin-associating proteinaceous substanceco-localizes with apoptin.
 14. The isolated or recombinantapoptin-associating proteinaceous substance of claim 12, wherein saidisolated or recombinant apoptin-associating proteinaceous substancebinds to the mouse transcription factor YY1.
 15. The isolated orrecombinant apoptin-associating proteinaceous substance of claim 12 thatcomprises amino acid positions 23 to 250 of SEQ ID NO:6.
 16. (canceled)17. An isolated or synthetic antibody that specifically recognizes anisolated or recombinant apoptin-associating proteinaceous substance orfunctional fragment thereof of claim
 12. 18. An isolated or recombinantapoptin-associating proteinaceous substance that induces apoptosis or afunctional part thereof that is specifically recognized by the antibodyof claim
 17. 19. A method of inducing apoptosis, said method comprising:contacting a susceptible cell with the isolated or recombinantapoptin-associating proteinaceous substance of claim 12, whereinapoptosis in said susceptible cell is induced.
 20. The method accordingto claim 19 wherein said apoptosis is p53-independent.
 21. The methodaccording to claim 19 further comprising contacting said susceptiblecell with an isolated or recombinant nucleic acid sequence encodingapoptin or fragment thereof or with apoptin or a functional fragmentthereof. 22.-26. (canceled)
 27. A method of detecting the presence ofcancer cells or cells that are cancer prone in a sample of cells, saidmethod comprising: transfecting cells in said sample with a nucleic acidsequence encoding an apoptin-associating proteinaceous substance,wherein said apoptin-associating proteinaceous substance comprises aminoacid positions 23 to 250 of SEQ ID NO: 6 or a functional fragmentthereof, which functional fragment is able to induce apoptosis and whichcomprises amino acids 54 to 248 of SEQ ID NO: 6; and determining thepercentage of apoptosis of cells in said sample, wherein a decrease inapoptosis as compared to normal cells is indicative of the presence ofcancer cells or cells that are cancer prone.
 28. A method of detectingthe presence of cancer cells or cells that are cancer prone in a sampleof cells, said method comprising: transfecting said cells in said samplewith a nucleic acid sequence encoding an apoptin-associatingproteinaceous substance, wherein said apoptin-associating proteinaceoussubstance comprises amino acid positions 23 to 250 of SEQ ID NO: 6 or afunctional fragment thereof, which functional fragment is able to induceapoptosis and which comprises amino acids 54 to 248 of SEQ ID NO: 6; anddetermining the intracellular localization of a proteinaceous substancederived from said isolated or recombinant nucleic acid sequence in cellsin said sample, wherein localization of said proteinaceous substance inthe nucleus is indicative of the presence of cancer cells or cells thatare cancer prone.
 29. The method according to claim 28, wherein thepresence of said proteinaceous substance in said cells is detected byimmunostaining said cells with an antibody.
 30. The method according toclaim 29, wherein said antibody comprises an antibody that specificallyrecognizes an apoptin-associating proteinaceous substance or functionalfragment thereof.
 31. A method of identifying a putative cancer-inducingagent, said method comprising: exposing a sample of cells to acancer-inducing agent; contacting said sample of cells with a nucleicacid sequence encoding an apoptin-associating proteinaceous substance,wherein said apoptin-associating proteinaceous substance comprises aminoacid positions 23 to 250 of SEQ ID NO: 6 or a functional fragmentthereof, which functional fragment is able to induce apoptosis and whichcomprises amino acids 54 to 248 of SEQ ID NO: 6; and detecting thepresence of cancer cells or cells that are cancer prone by determining apercentage of apoptosis of cells in said sample of cells, wherein saidpercentage is indicative of the carcinogenesis of said agent.
 32. Amethod of identifying a putative cancer-inducing agent, said methodcomprising: exposing a sample of cells to a putative cancer-inducingagent; contacting said sample of cells with the proteinaceous substanceof claim 12; and detecting the presence of cancer cells or cells thatare cancer prone by determining the intracellular localization of saidproteinaceous substance, wherein the presence of said cancer cells orcells that are cancer prone in said sample of cells is indicative of thecarcinogenesis of said agent.
 33. The method according to claim 31,wherein said putative cancer-inducing agent comprises a nucleic acidsequence.
 34. (canceled)
 35. A process for producing a peptidecomprising the peptide of SEQ ID NO:6, the process comprising:recombinantly expressing an isolated nucleic acid sequence encoding thepeptide of SEQ ID NO:6 in a cell.
 36. A peptide produced by the processof claim
 35. 37. (canceled)